JPS6236180B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Stool examination has been practiced in medicine since ancient times, but it is a diagnostic method that has acquired new practicality with the development of modern analytical methods.

In the case of a visual test, it already indicates the digestive function,
The consistency and shape of stool can indicate disease symptoms such as pancreatic insufficiency, steatorrhea, gastrointestinal bleeding, large intestine spasms, indigestion, and infectious diseases such as dysentery.
11th edition of Untersuchungsmethoden “Klinisches
Labor” p. 474, E. Merck, Darmstadt, 1970).

Furthermore, microscopic examination can provide another diagnostic value, for example for the presence of fat and muscle proteins in the stool. Additionally, the determination of the content of certain enzymes, parasites, bacteria, drugs and the like is also included within the scope of the test. However, testing the stool for latent blood is particularly important. The object of the invention and the various and thus disclosed possibilities for diagnosis, control tests and certification are explained in detail with respect to this method.

Enteric ulcer disease within the intestinal region is relatively frequent; intestinal ulcers are slow to develop and the disease is often recognized too late.

Although not completely obvious, a sign of the presence of an intestinal ulcer in the intestinal area is the appearance of blood in the stool.
However, blood in the stool is not detected, especially when only traces of blood are present in the stool and when the stool is darkly colored. Therefore, the development of a method capable of satisfactorily diagnosing even the smallest amount of blood in the stool would be a significant advance in identifying tumors within the intestinal region. The prerequisite is obviously to maintain a meat-free diet for eg 3 days before carrying out the test. The test follows the recognition of the blood component hemoglobin, more precisely the detection of the peroxidase activity inherent in hemoglobin.
For example, when acting on the components of guaiac resin,
At the same time, the presence of peroxides causes a clearly visible blue or blue-green coloration.

Similarly, other reagents that indicate the presence of hemoglobin by a color change can be used, such as benzidine, o-tolidine (dimethylbenzidine), tetramethylbenzidine, phenolphthalein, and the like.

Such tests are performed using impregnated paper strips or coated sheet strips and are commonly referred to as the dipstick method.

In practice, the test is often performed by first placing a small stool sample on a marked position on a test strip impregnated with a guaiac resin solution. Next, add a drop or two of hydrogen peroxide solution onto the test paper as a "developer". In this case, if blood is present, the blue or blue-green coloration described will occur.

Clinical experiments have shown that up to about 2 ml of blood in 100 g of excrement can be reliably detected even when the stool sample on the test strip is dry. However, when the developer was used immediately after placing the stool sample on the test paper, ie in a wet state, the detection limit was reduced to about 4 ml of blood in 100 g of feces.

The above-mentioned diagnostic method is subsequently referred to as the guaiac test method.

In terms of the diagnostic reliability of the test method, the reagents used and the finished test strips must undergo (static) quality control before they are delivered to the patient.

Furthermore, physicians and medical technical (in-house) assistants working with this diagnostic method should have means of continuous testing of the test method. Finally, it is extremely advantageous to be able to optionally display the test method.

The ability to test specimens under field conditions and express test methods with unambiguous results has heretofore been limited. If undiluted blood is applied for testing purposes onto the filter paper of a test strip prepared in the manner described above, the blood cells will probably not undergo hemolysis. in this case,
Peroxidase is not released and the guaiac reaction is likely to be atypical. Furthermore, even when blood hemolysis is taken into consideration, the conditions in the control test using blood are not very close to the conditions in the actual test.

Control test methods that can demonstrate a positive response to blood at lower detection limits are extremely powerful. Carrying out tests with blood (which has undergone hemolysis) is not very suitable for teaching methods. The closest approach to practice is to add a (predetermined) amount of blood to any stool sample whose blood content is therefore not determined from the beginning. In order to be able to make any statements about the quality of the test strips, a blood volume above the normal detection limit must be added to the stool sample. However, in the case of a positive reaction, the reliability of the test strip is proven beyond doubt only when in a parallel experiment it is confirmed that the stool before addition of blood (control test sample) gives a negative result, i.e. it is confirmed to be blood-free. be done. Nevertheless, if the control test sample without added blood responds, it should be ensured that there are no operational defects in the test strip or in handling. For this, it is likely that tests must first be carried out on various stool samples. Even with this method,
There is no absolute guarantee that the test strip will have detection sensitivity due to its specificity. The problem with such quality control tests is also clear from the following fact: Even a normal healthy person excretes approximately 0.5 to 1.5 ml of blood per stool in 24 hours; however, the amount of stool excreted each day Considering that the amount of stool varies between 100 and 500 g for individuals, the conclusion arises that it is simply impossible to define something like natural "normal stool" clinically.

It goes without saying that in the field of health, contact with feces should be limited to the absolutely necessary minimum for hygienic reasons. It can be inferred that diagnostic methods resulting from stool examinations are not always fully utilized due to the discomfort and risks associated therewith.
In this case, the difficulties already begin in teaching how to describe or analyze the stool types typical for diagnostic findings.

In the case of tests for latent blood, it is practically impossible to adjust the natural stool sample to a predetermined blood content, since, as already mentioned, in healthy normal individuals, variable amounts of blood are already present in the stool. This is because it is included in

matrices selected from cellulose, modified cellulose and other carbohydrates formed from water-refractory oxygen-containing solids, proteins, polyamides, (meth)acrylic acid derivatives as organic polymers or aluminum oxide as inorganic solids; Selected from the group of inorganic pigments with a brown and yellow hue, organic reactive dyes and powdered natural substances with a yellow to brown hue, suitable for producing a fecal-like coloration that adheres to the matrix material and does not run off. as a reference standard for the diagnostic detection and teaching of substances contained in stool, consisting of at least one colorant and a liquid selected from the group consisting of water, polyalcohols and alcohols as a lubricant; It has been found that the artificial stool used significantly satisfies the requirements for an artificial replacement for natural stool of the type produced. In particular, the artificial stool according to the present invention is suitable as a reference standard when detecting blood in stool by a test strip method, particularly a guaiac color test. Examples of poorly water-soluble oxygen-containing organic polymers used as matrix materials include: (α) cellulose and chemically modified cellulose derivatives or other carbohydrates, (β) carbohydrate materials according to (α) proteins such as gelatin, (γ) synthetic polyamides (PA 6.6) such as polyhexamethyleneadipamide, (δ) (meth)-acrylics such as polyacrylamide. derivative.

Examples of the poorly soluble oxygen-containing inorganic solid include aluminum oxide. Mixtures of various components of the type mentioned above also result in usable matrices.

Preferably particulate matrix materials and metal-free organic pigments with a particle size of up to approximately 300 μ, in particular in the range from 0.5 to 50 μ, are preferably used, with a hardness as similar as possible to natural faeces. The presence of large structural units, such as in natural stool, should not impair its usability. According to the invention,
Substitutes for the stool types that occur commonly and under pathological conditions can be prepared based on the above-mentioned components. The liquid content, such as water, preservative or lubricant, is selected depending on the stool type to be produced, for example. The content, of course, also depends on the water absorption and binding capacity of the host material. Generally, the water content is selected such that the spatula is usable.

If carbohydrates are used as matrix material according to (α), the liquid content is, for example, up to about 55% by weight, based on the total preparation, and in this case mainly water, lubricants, special glycerol or polyethylene, for example up to about 45% by weight. From 5 to 15% by weight of glycol and, for example, alcohol (ethanol) can be used as residual liquid. This ratio is also valid when replacing carbohydrate materials (partially) by proteins such as gelatin, for example by (β).

When using polyamides according to (γ), it is advantageous to work with relatively high contents of lubricant, for example 5 to 7 times the weight of glycerin (based on the polyamide).

Similarly, polyacrylamide-like (meth)
When using acrylic derivatives, relatively high liquid contents, for example 5 to 20 times, in particular about 18 times by weight, are advantageous, with the lubricant being present together with water, preferably in a ratio of 1:1 to 1:1.5. can be used.

Furthermore, when aluminum oxide is used as a sparingly soluble oxygen-containing organic matrix, the amount of liquid added is 2 to 2.5 times, especially about 2.3 times, and is generally about 12 to 2.3 times the amount of water.
A significant excess of lubricant of 18 times, especially 15 times, is recommended. Suitable lubricants include, inter alia, polyalcohols such as glycerin or polyethylene glycol (polywax 6000), optionally with the addition of monoalcohols such as ethanol.

Preservatives used are preferably food-acceptable preservatives, in particular solid preservatives such as sodium benzoate, benzoic acid or sorbic acid. However, for example the addition of ethanol can also have a preservative effect. The content of preservatives is determined by the effectiveness of the respective preservatives known, for example, from food preservation. The content generally does not exceed 3% by weight and is generally between 0.1 and 2% by weight, based on the total preparation.

By definition, the coloring agent to be used according to the invention is required to adhere to the matrix and not to run off, and to cause together with the matrix a permanent coloration as similar as possible to natural faeces. The adhesion requirements can, for example, be met in such a way that one or more colorants are combined with the matrix, for example in the form of a reactive dye, or the matrix can also be mixed with at least one insoluble colorant. , in which case adhesion is achieved by physical methods and runoff is likewise prevented.

The choice of colorant will therefore depend on the type of matrix and will also generally take into account the type and amount of coloration of the matrix material by known colorants. In addition, when selecting a colorant, consideration must be given to the fact that after treatment with a hydrogen peroxide solution as a "developer" there is no disturbing discoloration or a discoloration that would lead to a positive color reaction in the Guayama test. Nakatsuta.

Furthermore, the dye formed in the test must not be bound by the matrix itself.

Coloring agents include: (a) the class of yellow to brown pigments, for example mixtures based on iron oxides having brown and yellow hues, especially in a ratio of 3:2, or, for example, brown chromium iron; For example, mixed phase pigments mainly composed of iron and chromium oxide (Color index 1: pigmentbr-
aun29, color index2: No.77-500), (d) organic colorants, especially reactive dyes, which are bound to the matrix, e.g. heterounsaturated groups with reactive halogens, such as chlortriazine, -pyrazine, -pyrimidine; in which the halogen reacts with the OH groups or -NH ₂ groups of the substrate to be dyed (substituted dyes) (see "Textilveredlung", Vol. 7, p. 297, 1972) or with the formula: The coloring group of -SO ₂ -(CH ₂ ) ₂ -OSO ₃ Na is formed by -SO 2 -(CH 2 ) 2 -OSO 3 Na, and the resulting vinyl sulfone group reacts with the -OH group or -NH ₂ group of the substrate to be colored (addition Dye) (“Dtsch.Fa¨
rber-Kat.” Vol. 79, p. 188, 1975),
For example, the dye Remazol - Red B
(Reactive Red22 = color index number
14824), Remazol Gold Yellow G
(Reactive Yellow 17 = Color Index No. 18852), Remazol Brilliant Blue R (Reactive Blue 19 = Color Index No. 61200), especially in combination in a weight ratio of 2:2:1, (c) Natural with a yellow to brown hue Suitable finely distributable, especially powdered materials based on products, such as commercial cocoa powder.

Coloring can be carried out by separately coloring the matrix material or one of its components during the mixing of the components into the finished preparation or in particular before using the coloring agent according to (b). can.

In particular, an advantageous embodiment of the artificial stool according to the invention, which is suitable as a reference standard for the diagnostic detection of blood in stool by the dipstick method, comprises cellulose, modified cellulose and other carbohydrates, lubricants and the above-mentioned colors. It is obtained mainly from a preservative, a preservative, and water.
In particular, about 30-35% by weight of cellulose, especially about 33% by weight of cellulose and especially about water-soluble modified cellulose.
0.3-1.2% by weight, other water-binding carbohydrates, especially reticulated dextran 0.5-1.2% by weight, lubricants 5-10
% by weight, especially about 7.5% by weight of glycerin or polyethylene glycol, 1-2% by weight of colorants suitable to induce a color similar to natural stool, especially preservatives.
1.5 to 3% by weight, especially about 0.5 to 1% by weight of ethanol, 0.1 to 2% by weight of solid preservatives, especially about 1.5% by weight of sodium benzoate, benzoic acid or sorbic acid.
and preparations containing water to make up to 100% by weight are preferred.

Microcrystalline cellulose is used in particular as the cellulose component, water-soluble low-molecular cellulose ethers of the methyl hydroxyethyl cellulose type are used as the modified cellulose, and as reticulated dextran, in particular as a matrix for gel chromatography. A three-dimensionally reticulated swellable dextran is used, and suitable colorants for producing a fecal-like coloration include, in particular, the pigments mentioned above under (b), such as brown and yellow hues in a ratio of 3:2. Colorants based on iron oxides or reactive dyes as described in (c) are used.

With regard to the suitability of the preparation according to the invention as a control standard for the detection of latent blood by the dipstick method, the following should be noted in particular: When using pure cellulose-water-iron oxide mixtures However, the coating properties are only insufficient. Filter paper absorbs water from artificial stool very quickly and is extremely difficult to apply with stool.

A water-soluble low molecular weight cellulose ether of the methyl hydroxyethyl cellulose type is suitable for thickening artificial stool and significantly improves the applicability without impairing the dyeing reaction. The artificial stool according to the present invention is
A pasty consistency is obtained upon addition of the cellulose ether described above.

In contrast, polymeric cellulose ethers shift the detection limit of the guaiac test towards higher blood concentrations. Also, high concentrations of water-soluble cellulose ethers have similar results.

The addition of glycerin has a clear influence on the spreadability, on the one hand, and on the other hand, the glycerin material gives the same pale, shiny appearance as natural stool. The addition of reticulated dextran causes an improvement in the storage properties of the artificial stool due to the binding of excess water, and after 3 weeks of storage in vitro there is little tendency to form diluted fractions.

The production of artificial stool according to the present invention will be explained in detail with reference to the following example, but the present invention is not limited to this embodiment.

Example 1 5.5g of water-soluble low molecular weight methyl hydroxyethylcellulose (Tylose MH20) was added to undenatured ethanol.
Suspend in 8.9 g. After addition of 540 g of H ₂ O, stir for 15 minutes, then add 10.0 g of reticulated dextran (Sephadex G 25) for ion exchange purposes, and after 15 minutes add 14.5 g of sodium benzoate and 73.1 g of glycerin. The whole is then stirred or shaken for 3 hours. (When preparing stock solutions, it should be noted that Sephadex G25 will gradually precipitate. Shake before use.) In the dry state, microcrystalline cellulose (Avicel)
Thoroughly mix 333 g, brown iron oxide 9.0 g, and yellow iron oxide 6.0 g. Add the slightly cloudy solution prepared above to this and mix the two. After standing for 1 hour, the artificial stool is thoroughly mixed again.

Example 2 Standard stool with 0.4% blood by weight.

Human blood (preserved blood) is diluted with water at a ratio of 1:3 and left for 3 hours. Add 240μ of diluted blood to 20g of the artificial stool prepared in Example 1, and mix thoroughly. After that, leave it for about 1 hour,
Mix thoroughly again. Standard stools with approximately 0.4% blood should be used within 24 hours.

Example 3 Production of artificial stool in tubes Mix 0.52 g of water-soluble low molecular weight methyl hydroxyethyl cellulose (Tylose MH20) with 51 ml of water and leave for 24 hours with occasional stirring. Add to this 12g of glycerin DAB7 and 1.5g of sodium benzoate.
Add g. The solution thus obtained is added to 27 g of microcrystalline cellulose (Avicel PH101), previously intimately mixed with 0.8 g of brown iron oxide and 0.4 g of yellow iron oxide, and the mixture having a soft consistency is stirred.
1.5 diluted (1:3) human blood into the mixture
It can be added in a content of %. The stool thus obtained is highly suitable for handling in tubes.

Example 4 Proceed as in Example 1, but instead of soluble methylhydroxyethylcellulose gelatin (white,
Use 5.5g (extremely fine).

Example 5 Proceed as in Example 1, but instead of glycerin polyethylene glycol (Polywachs 6000) 73.1
Use g.

Example 6 The procedure is carried out as in Examples 1 to 3, but the iron oxide pigment is replaced by a reactive dye which is bound to the dextran. Colored dextran ingredient is dextran 20g
Remazol Brilliant Blue (color index number 61200) 100mg, Remazol Red B (color index number 14824) 200mg and Remazol Gold Yellow G (color index number
18852) is stirred into 200 ml of distilled water with 200 mg of the colorant mixture, and after adjusting the pH value to 12.0, it is incubated at room temperature for 16 hours. After the end of the incubation, it is precipitated with ethanol and washed several times. The product has a brown color after drying.

Example 7 Artificial faecal microcrystalline cellulose (Avicel) based on colored cellulose is colored brown as in Example 6.

2g of colored microcrystalline cellulose and glycerin
Mix 3.6g of DAB 7. A dark brown product is obtained with satisfactory coating properties.

Example 8 Artificial stool based on colored dextran.

Reticulated dextran (Sephadex G 25) for ion exchange purposes is colored as in Example 6.

Analogously to Example 1, from the components listed therein, but monochromatically colored dextran was used without the addition of pigments, microcrystalline cellulose (333
(300g instead of g) from the beginning to obtain artificial stool with a relatively bright hue.

Example 9 Artificial stool based on colored polyamide.

The polyamide is colored brown with Remazol colorant as in Example 6. Mix 15.0 g of glycerin DAB7 with 662.5 g of colored polyamide. A product with good stability is obtained with optimum application ability.

Example 10 Artificial stool based on polyacrylamide + iron oxide pigments Polyacrylamide (Biogel P 150) 2.5 g with the addition of 0.9 g of brown iron oxide and 0.6 g of yellow iron oxide
20.0 g of glycerin and 25.0 g of distilled water are mixed with g. Although this synthetic stool is suitable for illustrative purposes, it exhibits only poor spreading ability due to the swollen particles.

Example 11 An artificial stool based on polyacrylamide + dextran colored with a reactive dye can be prepared as in Example 10, but with the addition of 1.5 g of colored dextran according to Example 6 instead of the iron oxide pigment. can.

Example 12 Artificial stool based on aluminum oxide + iron oxide pigment 14.8 g of aluminum oxide (Martoxin) is mixed with 2.0 g of distilled water and 30.0 g of glycerin DAB7 with the addition of 0.9 g of brown iron oxide and 0.6 g of yellow iron oxide. do. This artificial stool exhibits the sticky consistency of a relatively dilute liquid.

Example 13 Artificial stool based on cellulose with cocoa powder as colorant 20 g of microcrystalline cellulose (merk, for thin layer chromatography), 20 g of cocoa powder and distilled water.
Mix 200ml. Process the mixture on a roll mixer overnight. It is then filtered with suction through a frit and washed with water. Mix 5 g of glycerin DAB7 with 7 g of the wet product.

Use of the preparations according to Examples 1 to 13 By adding about 1.5% by weight of blood to the preparations according to Examples 1 to 13, it is generally possible to detect blood in the stool diagnostically by the dipstick method, in particular the guaiac test. Provides a useful reference standard for detection.

The detection sensitivity of blood in artificial stool according to Example 1 is 0.4% by weight of blood when commercially available test strips are used.

The detection limit of blood in natural stool is estimated to be about 1.2-1.6% by weight of blood under the same conditions. Therefore, the detection limit of blood in artificial stool according to the present invention is higher than that of blood in natural stool. below this limit. That is, one important prerequisite for using the artificial stool according to the invention for control purposes is satisfied. The freshly prepared standard stool according to Example 2 is therefore suitable for, for example, a test strip control test.

Claims (1)

[Claims] 1. Cellulose, modified cellulose and other carbohydrates formed from oxygen-containing solids that are poorly soluble in water, proteins, polyamides, (meth)acrylic acid derivatives as organic polymers or oxidized as inorganic solids. Matrix selected from aluminum,
Selected from the group of inorganic pigments with a brown and yellow hue, organic reactive dyes and powdered natural substances with a yellow to brown hue, suitable for producing a fecal-like coloration that adheres to the matrix material and does not run off. as a reference standard for the diagnostic detection and teaching of substances contained in stool, consisting of at least one colorant and a liquid selected from the group consisting of water, polyalcohols and alcohols as a lubricant; Artificial stool used. 2 The matrix is at least partially about 300μ
The artificial stool according to claim 1, comprising particulate matter having a particle size within the range of . 3. Use protein instead of modified cellulose.
Artificial stool according to claim 1. 4. From claim 1, in which a coloring agent suitable for producing a fecal-like coloration that sticks to the matrix material and does not run off is used in an amount of 0.5 to 5% by weight, based on the total preparation. Artificial stool as described in any one of paragraphs up to paragraph 3. 5. Claims in which the coloring agent consists of a pigment mixture based on iron oxide or the metal-free organic pigment brown chromium iron (color index number 77-500), in particular with a brown and yellow hue in a ratio of 3:2 1st
Artificial stool as described in section. 6. The artificial stool according to claim 1, wherein the coloring agent is covalently bonded to the matrix via a vinyl sulfone group. 7 The colorant is a combination of the dyes Remazol Red B (Color Index No. 14824), Remasol Gold Yellow G (Color Index No. 18852) and Remazol-Brilliant Blue R (Color Index No. 61200) in a ratio of 2:2:1. The artificial stool according to claim 6, comprising: 8. The artificial stool according to claim 1, wherein the coloring agent comprises cocoa powder. 9. The artificial stool according to claim 1, which uses polyethylene glycol as a lubricant. 10. The artificial stool according to claim 1, which contains a preservative. 11. The artificial stool according to claim 10, wherein one or more food-grade preservatives are used as preservatives. 12 30 to 35% by weight of cellulose, 0.3 to 1.2% by weight of water-soluble modified cellulose, 0.5 to 1.2% by weight of other carbohydrates, 5 to 15% by weight of lubricant, and a coloring agent suitable for inducing coloring similar to natural stool. 2% by weight, up to 3% by weight of preservatives (including 0.5-1% by weight of ethanol and 0.1-2% by weight of solid preservatives) and 100% by weight of preservatives.
Artificial stool according to claim 1 or 2 and any one of claims 4 to 11, consisting of water to make up to % by weight. 13 30-35% by weight of crystalline cellulose, 0.3-1.2% by weight of water-soluble low-molecular cellulose ether, 0.5-1.2% by weight of three-dimensionally reticulated swellable dextran, 5-10% by weight of glycerin, in a ratio of 3:2. A mixture of metal-free organic pigments based on iron oxide with a brown or yellow hue or the metal-free organic pigment brown chromium iron or the colorants Remazol Red B, Remazol Gold Yellow G and Remazol Brilliant Blue R in a 2:2:1 ratio. 1 to 2% by weight of a mixture comprising a combination of ratios of 1 to 2% by weight of ethanol, 0.5 to 1% by weight of ethanol and 0.1 to 2% of water to make up to 0.1 to 2% by weight of a solid preservative also suitable for food grade.
Artificial stool described in Section 2. 14. Claim 1, wherein the preparation contains synthetic polyamide and lubricant in a weight ratio of 1:5 to 1:7.
The artificial stool according to paragraph 2, paragraph 4, or paragraph 9. 15. The preparation contains polyacrylamide together with 5 to 20 times the amount of liquid, where the liquid consists of water and lubricant in a ratio of 1:1 to 1:1.5. The artificial stool described in paragraph 2, paragraph 4, or paragraph 9. 16 If the preparation contains twice as much aluminum oxide or
Water to lubricant ratio 1:12 ~ with 2.5 times liquid
Claims 1 and 2 containing at a ratio of 1:18
Artificial stool according to paragraph 5, paragraph 8 or paragraph 9. 17. The artificial stool according to claim 1, which is used as a reference standard when blood in stool is diagnostically detected by the test strip method.